While many investigations utilizing amphibian, avian and mammalian tissue have suggested that the pigment epithelium (PE) is necessary for outer segment membrane production of normal quantity and structure, the exact mechanism(s) by which this occurs is not known. The PE may serve one or more functional supports to the underlying photoreceptor cells, i.e.,the appropriate microenvironment, a structural template via the interphotoreceptor matrix or be the source of some trophic factor(s) that regulate gene expression. In this proposal, the role of the PE on photoreceptor differentiation and outer segment membrane organization will be systematically explored using an in vitro model in which retinas will be removed to culture in an undifferentiated state. The retinas mature and elaborate outer segments in both the presence and absence of the PE, however the membranes are disorganized in its absence. Because photoreceptors do produce outer segment membrane in the absence of the PE the system can be exploited to determine what factor(s) are able to alter the organization of the membranes and the synthesis of the proteins therein. Opsin and interphotoreceptor retinoid binding protein (IRBP) gene expression and protein synthesis as well as outer segment organization will be compared and contrasted in retinas allowed to mature without a PE vs. without a PE yet in the presence of various potential differentiation factors including lactose, growth factors, retinoids and IRBP. The PE undoubtedly plays a number of complex roles during the development of photoreceptors. It is unlikely that any one of the components when tested alone would restore a full level of expression and synthesis of the photoreceptor specific differentiation markers. However, a systematic assessment of the ability of each of the test conditions to enhance expression and synthesis above control levels offers a direct approach to identifying the various components that could be provided by the PE which support photoreceptor differentiation.